The ecdysteroid UDP-glucosyltransferase ( egt ) gene of Mamestra brassicae multinucleocapsid nucleopolyhedrovirus (MbMNPV) has been cloned and characterized. MbMNPV egt potentially encodes a protein of 528 amino acids. Analysis of the substrate specificity of the MbMNPV EGT protein showed that it mirrors that of Autographa californica MNPV (AcMNPV) EGT. MbMNPV EGT also appears to be secreted from infected cells. Confirmation that the cloned gene encodes an active EGT was obtained by transient expression assays. Phylogenetic trees of NPVs were generated based on the alignment of baculovirus EGT sequences. These phylogenies support the classification of MbMNPV as a group II NPV that is most closely related to Spodoptera exigua MNPV. Comparison of the EGT-based phylogenies with polyhedrin/granulin-based phylogenies shows that the position of AcMNPV is different in the two trees, possibly indicating that AcMNPV acquired its polyhedrin gene by recombination with another virus.